This research is designed to examine the salient structural, mechanistic and regulatory features of bovine angiotensin converting enzyme when bound to the endothelial plasma membrane. This work will be performed in two stages. The first stage will be the examination of the homogeneous solubilized enzyme. A basic physio-chemical characterization of the enzyme will be performed and will include the determination of its molecular weight, amino acid and carbohydrate composition, metal content, amino and carboxy terminal amino acid sequences, extinction coefficient of alpha-helical and beta-sheet structure. Limited trypsinolysis of the enzyme will be used to examine its overall three dimensional structure and to localize the metal ion binding site and the attachment sites for its carbohydrate substituents. The hydrolysis of a series of hippuryl dipeptides will be studied to determine the relationship between both substrate structure and viability and substrate structure and Cl- ion requirement for hydrolysis. Equilibrium dialysis will be used to determine if the enzyme contains a unique Cl- ion binding site, and if it does, the nature of this site will be determined by chemical modification. The role of the metal ion in substrate hydrolysis will be examined by comparison of the kinetics of substrate hydrolysis for native and reconstituted cobalt enzymes. Equilibrium dialysis studies using the holoenzyme will be performed to determine if the metal ion is necessary for Cl- ion binding. As the final study of stage one, a steady-state kinetic analysis of substrate hydrolysis will be performed to determine the kinetic mechanism of hydrolysis and Cl- activation. Upon completion of these studies, stage two will be begun which is to characterize these properties of the enzyme in isolated purified endothelial plasma membrane. The relationship between substrate structure and viability, the role of the metal ion in substrate hydrolyse, the relationship between Cl- ion requirement and substrate structure and the steady-state kinetic mechanism will all be determined for the membrane bound enzyme.